Imidazolium salt’s toxic effects in larvae and cells of Aedes aegypti and Aedes albopictus (Diptera: Culicidae)

Aedes aegypti and Aedes albopictus are the main vectors of arboviruses such as Dengue, Chikungunya and Zika, causing a major impact on global economic and public health. The main way to prevent these diseases is vector control, which is carried out through physical and biological methods, in addition to environmental management. Although chemical insecticides are the most effective strategy, they present some problems such as vector resistance and ecotoxicity. Recent research highlights the potential of the imidazolium salt "1-methyl-3-octadecylimidazolium chloride" (C18MImCl) as an innovative and environmentally friendly solution against Ae. aegypti. Despite its promising larvicidal activity, the mode of action of C18MImCl in mosquito cells and tissues remains unknown. This study aimed to investigate its impacts on Ae. aegypti larvae and three cell lines of Ae. aegypti and Ae. albopictus, comparing the cellular effects with those on human cells. Cell viability assays and histopathological analyses of treated larvae were conducted. Results revealed the imidazolium salt’s high selectivity (> 254) for mosquito cells over human cells. After salt ingestion, the mechanism of larval death involves toxic effects on midgut cells. This research marks the first description of an imidazolium salt's action on mosquito cells and midgut tissues, showcasing its potential for the development of a selective and sustainable strategy for vector control.

and spatial spraying in comparison to larvicidal compounds 8 .Meier et al. 9 asserted that employing chemical or biological products against larvae results in a reduction in the number of adult insects, leading to inhibition of reproduction.Because of their widespread use, the selective pressure on the neuronal targets of insect populations has led to the emergence of resistant populations 10,11 .Consequently, the efficacy of some insecticides has been compromised, which led to their discontinuation 9 .Alternatively, the adoption of the "pesticide treadmill" strategy, involving a mixture of several pesticides or a high-dosage regimen with predefined rotation, has been implemented 12,13 .
Significantly, even after years of insecticide application, their presence endures in the ecosystem 14 .Meier et al. 9 provided an analysis of the ecological cost of chemical products and highlighted the excessive use and the need for higher doses to control insects.The excessive application not only damages the ecosystem but also results in environmental accumulation.Such profound use of these compounds contributes to severe environmental harm and has implications for human health 15,16 .Moreover, sustained application over time has revealed adverse effects such as water contamination, toxicity to non-target organisms, and environmental pollution 17,18 .
Hence, there is a crucial emphasis on the pursuit of novel pest control methods that minimally impact the environment while effectively managing mosquito vectors 3 .The potential toxicity of synthetic chemicals to non-target organisms 19 makes it very difficult to develop new and more selective compounds for target insects 20 .Consequently, there is a growing interest in the study and development of "green" molecules for the control of mosquito vectors 21 .
Various imidazolium salts (IS) with biological activities, such as antimicrobial, antifungal, antitumor, and anti-inflammatory, have been identified 22 .Some studies have demonstrated the effective larvicidal action of 1-methyl-3-octadecylimidazolium chloride (C 18 MImCl) in controlling Ae. aegypti 23,24 and Culex quinquefasciatus 25 .Pilz-Júnior et al. 25 also confirmed the safety of using this IS through acute oral toxicity in Wistar rats by the up and down test (OECD 425), as well as phytotoxicity assays on Lactuca sativa.
In the present study, we demonstrate that IS C 18 MImCl has high selectivity for cells of Ae. aegypti and Ae.albopictus compared to human fibroblasts.Larvae of Ae. aegypti treated with the same salt presented cellular changes in the midgut, with modification from columnar to squamous tissue, the presence of a large number of vacuoles, and loss of microvilli from epithelial cells.

Cytotoxicity assays
Cytotoxicity assays demonstrated that C 18 MImCl promoted a toxic effect on mosquito cell lines.In relation to the negative control group (culture medium only), treatment for 48 h with a concentration of 0.027 µM caused a decrease of 72.8, 80.3 and 50.2% in the number of cells for Aag2, C6/36 and U4.4,respectively (Fig. 1).In general, there was a dose-dependent effect on the three cell lines of Aedes spp.treated with the IS C 18 MImCl (Fig. 2).For the C6/36 and U4.4 strains, there were significant differences (p < 0.0001) between doses and time of exposure (24, 48 and 72 h), and the longer the time, lower doses were needed to promote a toxic effect.The Aag2 strain did not show differences between exposure times (p = 0.0125).As with the three cell lines of Aedes spp., fibroblasts also showed a dose-dependent response (p < 0.0001).For the cell lines of Aedes spp.and fibroblasts, the toxic concentrations ranged from 0.00027 to 2.7 µM and 0.5 to 20 µM, respectively (Table 1).The raw data is available as supplementary material.
The IC 50 values calculated for the C6/36, U4.4, and Aag2 strains from the treatments with the concentrations of 0.00027, 0.0027, 0.027, 0.27 and 2.7 µM during 24, 48 and 72 h are described in Table 2.Those IC 50 values for fibroblasts treated with 0.5, 1.0, 2.5, 5.0, 7.5, 10 and 20 µM for 48 h are given in Table 3, together with the selectivity indexes calculated from these IC 50 values for each cell type (Tables 2, 3).

Histopathology
The behavior of Ae. aegypti larvae, whether treated or untreated with IS, showed differences in terms of toxicity.Untreated larvae displayed typical mobility in the aquatic environment, characterized by sinuous swimming movements at various depths within the container.Histological sections depicted a normal appearance in the midgut, foregut, and hindgut, peritrophic membrane, muscles, brush border, fat body, and epithelial cells (Figs. 3A and 4A).Contrastingly, larvae treated with IS showed signs of toxicity, including reduced mobility, altered movements, and decreased response to stimuli, aligned with the criteria for evaluating larvicidal products established by the World Health Organization 26 .Histopathology analyses revealed significant changes in the midgut epithelium, characterized by a loss of cell polarity, switching from columnar to a round shape, intense vacuolization, cell disorganization, loss of cell-cell adhesion, and loss of microvilli.Damage was observed in the fat body tissue, accompanied with larval atrophy and variations in the dimensions of the treated larvae, with the integument being closer to the intestinal epithelium (Figs.3B and 4B).

Discussion
The findings of this study illustrate a significant selectivity of C 18 MImCl for Aedes spp., as evidenced by the inhibitory concentrations for each cell type.In comparison to human fibroblasts, the dosage necessary to achieve an equivalent toxic effect in the C6/36 lineage must be 1049 times higher.Similarly, concerning the U4.4 and Aag2 cells, the doses necessary to induce a comparable toxic effect in human fibroblasts must be 254 and 619 times higher, respectively.
Until now, the research that explored the impact of IS on different cell types is limited.Martins et al. 27 investigated the toxic effect of different IS molecules against Leishmania amazonensis and certain human cells.In peripheral blood mononuclear cells (PBMC), almost all IS caused dose-dependent cytotoxicity.Assessing the CC 50 PBMC / IC50 Leishmania ratio, C 18 MImCl showed low cytotoxicity for PBMC with a selectivity index of 15.35.The authors also performed a hemolytic assay, demonstrating that human erythrocytes were less susceptible to the cytotoxic effect of IS, with significant hemolysis observed only at concentrations ≥ 30 μM, yielding a selectivity index ≥ 30 27 .
The work conducted by Pilz-Júnior et al. 25 also highlighted the low cytotoxicity of C 18 MImCl, revealing the effectiveness of IS in controlling Culex quinquefasciatus larvae with minimal impact on non-target-organisms.In the V79 cell line (murine fibroblasts), C 18 MImCl demonstrated biocompatibility even at doses up to 5 µM, with a CC 50 of 12.8 µM.For HaCat cells (keratinocytes), the estimated CC 50 for C 18 MImCl was 18.1 µM 25 .Additionally, Pilz-Junior et al. 24 demonstrated bacterial modification in the microbiota of larvae treated with the same IS.
In this study involving Ae. aegypti treated with C 18 MImCl, various toxic effects were observed.These included alterations in the midgut epithelium, featuring a transition from columnar to squamous tissue, the presence of vacuoles, and loss of microvilli in the epithelial cell layer.As far as we are aware, this is the first study demonstrating the histopathological effect an IS on mosquito larvae.Interestingly, these toxic effects are comparable with those found in larval tissues treated with bacteria and fungi 20,28,29 .These effects differed significantly from those observed when mosquito's species were treated with chemical insecticides like the organophosphate temephos.Several studies utilizing natural bioactive compounds have demonstrated similar alterations in midgut cells like the IS studied 30 .
Histological analysis of Ae. aegypti and Anopheles stephensi treated with polysaccharides from Bacillus licheniformis revealed muscle damage, abdominal atrophy, and lesions in the midgut 28 .Treatment with a limonoid compound from Penicillium oxalicum induced damage to microvilli, peritrophic membranes, and epithelial cells of C. quinquefasciatus larvae 29 .In An. stephensi larvae treated with fraction 1 (F1-emodin) of the mycelial extract of Aspergillus terreus, the midgut emerged as the most affected tissue, displaying disruption in the peritrophic membrane and the intestinal epithelium.Additionally, cellular vacuolization, muscle damage, and disorganization of the brush border were observed.conducted a histopathological analysis of C. quinquefasciatus larvae exposed to Beauveria bassiana-28 extract.Their results similarly demonstrated damage to the cells of the intestinal epithelium, accompanied by vacuolation, destruction of the cell membrane, infiltration of the luminal contents into the muscle tissue and cell damage.Additionally, disorganization of the fat body was observed.
The lesions observed in the histopathological analysis of this study suggest that the IS-induced larval death process is linked to the disruption of intestinal homeostasis, potentially leading to various death pathways: (a) interference in nutrient absorption due to the loss of microvilli; (b) damage to the intestinal cell membranes, enabling infiltration of luminal contents into the hemocoel, which may result in (b-1) septicemia, (b-2) damage to muscle cells causing paralysis of movement, or (b-3) entry of IS molecules into the fat body, destabilizing trophocytes and oenocytes and interference in larval metabolism.
It is noteworthy that there is no class of chemical larvicide with the properties of the presented imidazolium salt, which does not present phytotoxicity or toxicity to mammals 25 .As described in the work of Goellner et al. 23 , C 18 MImCl showed a residual effect with 95% and 80% larval mortality for 36 and 78 days, respectively, after being dried under environmental conditions for at least two months and then dissolved in water for use.
In conclusion, the IS C 18 MImCl exhibits notable selectivity for Ae.aegypti and Ae.albopictus cells in comparison to human fibroblasts.The mechanism of death appears to be associated with toxic effects of C 18 MImCl in larvae's midgut after its ingestion from water.These findings underscore the potential of this IS as a promising agent for addressing the current challenges in Aedes control.Additionally, the importance of conducting further investigations with diverse approaches is emphasized to develop new strategies for effective vector control.

Larvae, reagents, and cell culture
The larvae of Ae. aegypti (Rockefeller strain) come from a colony maintained under controlled conditions of temperature (± 28 ºC) and humidity (± 80%) in the Laboratory of Arthropod Vectors of the Department of Microbiology, Immunology, and Parasitology at Institute of Basic Health Sciences-ICBS/UFRGS.
Cell line of Aedes aegypti Aag2 -derived from embryonic tissue 32,33 and Ae.albopictus U4.4 -derived from newly hatched axenic larvae 34 , were received from the University of Brasilia.Aedes albopictus C6/36 ATCC® CRL-1660™ -derived from newly hatched axenic larvae 34,35 was obtained from the Cell Bank of Rio de Janeiro, Brazil.Two different Ae.albopictus cell lines were used to evaluate the effectiveness of C 18 MImCl and verify whether the same effect occurred in both strains, and thus provide robustness for the analysis and interpretation of the results presented.Primary fibroblasts were donated by the Laboratory of Cell Biology of the Institute of Basic Health Sciences at UFRGS.All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM -Gibco, Thermo Fischer Scientific, Massachusetts, USA) with low levels of glucose, supplemented with 1% non-essential amino acids (NEAA -Gibco, Thermo Fischer Scientific, Massachusetts, USA), maintained at 28 °C in a 5% CO 2 incubator.Primary human fibroblasts were cultured in DMEM medium at 37 °C and maintained in a 5% CO 2 incubator.

C 18 MImCl activity in human and mosquito cells
Cell viability was determined by measuring protein content with the sulforhodamine B assay (SRB) 36 .Mosquito cell lines (6 × 10 3 cells/well) and human fibroblasts (2 × 10 3 cells/well) were incubated overnight in DMEM culture medium.The IS C 18 MImCl was diluted in ultrapure water (Milli-Q) and treatments were performed at concentrations ranging from 0.00027 to 2.7 µM for 24, 48, and 72 h for cell lines of Aedes spp., and concentrations between 0.5 to 20 µM for 48 h for fibroblasts.Hydrogen peroxide (10 mM) was used as a positive control.After the incubation period, the cell monolayers were fixed with 10% trichloroacetic acid, washed with running water, and kept overnight at room temperature for drying.Afterward, staining was performed for 30 min with 0.4% SRB dye diluted in 1% acetic acid, excess dye was removed by repeated washing with 1% acetic acid.Protein-bound dye was dissolved in 10 mM Tris base solution for optical density (OD) determination.Next, absorbance was read at a wavelength of 560 nm in a Multiskan GO microplate reader (Thermo Fisher Scientific).
The concentration of IS that caused the death of 50% of cells was determined by nonlinear regression analysis (curve fit; hill slope = 1; F = 50) at confidence level of 95%, using the equation: Treatment = Log (treatment) vs. response-Find ECanything Least squares fit.For cell lines of Aedes spp., IC 50 values were calculated at 24, 48, and 72 h of exposure, the one for fibroblasts at 48 h.The selectivity index (SI) of C 18 MImCl was determined by the equation: IC 50 against fibroblasts/IC 50 for Aedes spp.cells.

Histopathological analysis of larvae exposed to C 18 MImCl
The histopathological analysis was performed according to Lemos et al. 37 .Two groups of larvae were used in this bioassay: 25 larvae of the 3rd and 4th instars of Ae. aegypti maintained only in 150 mL of distilled water (control group), and 25 larvae treated with 150 ml of C 18 MImCl at CL 99 -18.862µg/ml 23 .The 12-h treatment time was determined from signs of toxicity observed during the exposure of the groups, mainly, slowness and change in movements, and decreased response to stimuli 26 .After exposure, the samples were fixed in Karnovsky's solution 38 for 24 h at 4 °C, and washed with 70% alcohol.Dehydration was performed by immersing larvae in a series of increasing concentrations of ethanol: 70% 10 min, 95% 15 min, 100% 20 min, and 100% 30 min.Then the samples were soaked in a resin solution (Leica Historesin Embedding Kit) at room temperature.Embedding was performed in 6 × 8 mm polyethylene molds.The molds were kept at 40 °C for at least 12 h to ensure complete polymerization of the resin.Longitudinal sections of 3 μm were made using a Lupetec MRP 2015 semi-automatic rotary microtome with disposable blades.For each resin block, 10 to 15 microscopy slides were prepared, on which six sequential sections were fixed.These slides were stored in an oven at 30 ± 2 °C for 24 h.For the analysis, slides were stained with Harris hematoxylin and eosin.The sections were examined and photographed using a Nikon Eclipse Si microscope coupled with a Digital Sight 1000 camera in conjunction with the Capture 2.3 program.

Statistical analysis
Viability assays were performed in quadruplicate and repeated three times.Data were submitted to the Kolmogorow-Smirnow test to assess normality.Variables with normal distribution were compared by analysis of variance, repeated measures or one-way ANOVA, and Tukey's multiple comparisons test.Significant differences were considered when p value < 0.0001.The GraphPad Prism 8.3.0 (GraphPad Software, Boston, Massachusetts, USA) program was used to carry out the analyses.

Figure 1 .
Figure 1.Photomicrographs showing the effect of the imidazolium salt C 18 MImCl on the three mosquito cell lines, treated for 48 h with a concentration of 0.027 µM.It is possible to observe a decrease in the number of cells in the treated group compared to the control group (culture medium only).Indicating less cell proliferation after treatment.Magnification 10×.

Figure 2 .
Figure 2. Viability assay of cells treated with C 18 MImCl.C6/36, U4.4, and Aag2 cell lines were treated with different concentrations of C 18 MImCl, and H 2 O 2 10 mM, and cell viability analyzed at 24, 48 and 72 h using the SRB assay.Primary human fibroblasts were treated and analyzed at 48 h.Percentage to the negative control (100%).Mean with range.

Figure 3 .
Figure 3. Histological analysis of internal tissues of Ae. aegypti larvae treated and untreated with C 18 MImCl.(A) Light micrograph of a negative control larva; (B) light micrograph of a larva treated for 12 h with LC 99 of C 18 MImCl.The sections depicted a normal appearance in the midgut, foregut, and hindgut, peritrophic membrane, muscles, brush border, fat body, and epithelial cells.EC = Intestinal epithelial cells; L = Lumen; F = Food; T = Tegument; FB = Fat body.Hematoxylin-eosin.Magnification 10×.

Figure 4 .
Figure 4. Histological analysis of internal tissues of Ae. aegypti larvae treated and untreated with C 18 MImCl.(A) Light micrograph of a negative control larva; (B) light micrograph of a larva treated for 12 h with LC 99 of C 18 MImCl.The analyses revealed significant changes in the midgut epithelium, characterized by a loss of cell polarity, switching from columnar to a round shape, intense vacuolization, cell disorganization, loss of cell-cell adhesion, and loss of microvilli.Damage was observed in the fat body tissue, accompanied with larval atrophy and variations in the dimensions of the treated larvae, with the integument being closer to the intestinal epithelium.EC = intestinal epithelial cells; L = Lumen; F = Food; T = Tegument; FB = Fat body; PM = Peritrophic membrane; BB = Brush edge; N = Core.Hematoxylin-eosin.Magnification 40×.

Table 1 .
Statistical analysis for the cell lines of Ae. aegypti and Ae.albopictus treated with C 18 MImCl for 24, 48, and 72 h, as well as human fibroblasts treated for 48 h.Percentage of the negative control (100%).DF = Degrees of freedom.

Table 2 .
IC 50 values calculated for Ae.aegypti and Ae.albopictus cell lines During 24, 48, and 72 h of treatment with C 18 MImCl.*Doses presented in µM.**Within 95% confidence intervals, ECF: Confidence interval where the required effective concentration (EC) can be found.

Table 3 .
IC 50 values calculated for fibroblasts after 48 h of treatment with C 18 MImCl, and selectivity indexes (SI) between mosquito cells and human fibroblasts.*Doses presented in µM.**Within 95% confidence intervals, ECF: Confidence interval where the required effective concentration (EC) can be found.